人工模拟动态微环境用于膀胱肿瘤细胞有氧糖酵解代谢机制的研究

The reason for high aerobic glycolysis in cancers is still unknown to scientists, and mitochondria dysfunction is considered as the direct cause. However, recent researches have shown that many factors and pathways are involved in this process, such as acidic microenvironment, highly expressive pyruvate kinase M2 (PKM2) and other signal pathways. The conventional research techniques in cancer have some limitations that they couldn't be used in dynamically changing environment to investigate complex aerobic glycolysis. To resolve it, we utilize precisely controlled microfluidic technology, and look at the bladder cancer metabolism and its role in cancer proliferation over dynamically changing environment. To our knowledge, this multi-disciplinary research strategy in bladder cancer is the first time. This project includes the following three major components: ① Developing a microfluidic platform that can subject a population of bladder cancer cell lines (T24 and 5637) to a continuously varying supply of glucose and oxygen medium to find the relation between periodic changing energy source and aerobic glycolysis conditions; ② Determining the metabolites such as pyruvate, lactate, PKM2 and nicotinamide adenine dinucleotide phosphate (NADPH), which are produced in glycolysis pathway and pentose phosphate pathway; ③ Investigating the functions of the key glycolytic regulator PKM2 in metabolism and cancer progression and its emerging role as a potential target for cancer therapy. The aim of this project is to clarify the metabolic mechanism for why do cancers have high aerobic glycolysis, to investigate the functions of PKM2 in metabolism and cancer progression, and to provide a new vision and novel target for cancer research and therapy.

肿瘤细胞有氧糖酵解及其在肿瘤发生中的确切作用机理尚未阐明，多认为线粒体功能缺失是直接原因。最新研究表明酸化的微环境、高表达的丙酮酸激酶M2（PKM2）和其它信号通路等参与作用。鉴于肿瘤细胞糖代谢的复杂机制，常规肿瘤学方法较难在连续动态环境下对其进行观察和研究。因此，本项目用微流控芯片精确操控及微量输入技术，动态研究膀胱肿瘤代谢异常及其在肿瘤发生发展中的作用机制。目前，尚未有相同研究报道。 本项目①通过在微流控芯片上输入周期性变化的葡萄糖碳源及氧源，人工动态模拟细胞生长微环境，实时观察膀胱肿瘤细胞（T24和5637）的代谢异常；②结合常规肿瘤学方法，测定糖酵解和磷酸戊糖途径代谢产物丙酮酸、乳酸、PKM2和NADPH（还原型辅酶II）等指标；③阐明肿瘤细胞高度依赖有氧糖酵解获能的代谢机制和其作用机理，发掘PKM2的角色功能和其作为肿瘤靶向治疗调控元件的潜能，为肿瘤防治提供新的视角和靶点。

正常细胞生长所需能量主要来自糖代谢，通过将葡萄糖氧化为二氧化碳和水以获得所需三磷酸腺苷（ATP），而肿瘤细胞则将葡萄糖代谢为乳酸以获得快速生长，糖代谢异常是肿瘤细胞的一个重要特征，肿瘤细胞糖代谢呈现以有氧糖酵解为主的特殊形式，也是近年来肿瘤学研究的热点和未解难题之一。正常细胞在有氧条件时是通过有氧氧化来分解葡萄糖获取能量的，只有在低氧时才止步于糖酵解。而肿瘤细胞无论氧气存在与否都主要依赖糖酵解进行代谢，消耗大量葡萄糖并产生乳酸，这一现象称为Warburg 效应。肿瘤出现Warburg 效应可能与肿瘤的低氧微环境、糖酵解关键酶活性增加和同工酶谱的改变、糖异生关键酶活性的降低以及某些信号通路异常有关。本研究通过在微流控芯片上培养膀胱癌细胞系T24，体外模拟肿瘤细胞生长微环境，并测定其代谢产物尼克酰胺腺嘌呤二核苷酸磷酸（NADPH）等的含量变化。在此基础上，本项目也尝试应用传统中医学对肿瘤的认识，发展出了膀胱癌的病因病机和有氧糖酵解代谢之间的内在联系，并提出了理论假说 “膀胱癌湿热瘀毒与其有氧糖酵解代谢异常相关”。同时，利用传统抗癌中草药白花蛇舌草和半枝莲，作用于膀胱癌细胞，发现其通过降低表达miR-155，从而降低Akt的磷酸化水平，而保持Akt信号通路缄默，参与诱导肿瘤细胞凋亡。